Celastrol pretreatment has been shown to protect against myocardial ischemia/reperfusion (I/R) injury, but the underlying mechanism is poorly understood. This study aimed to investigate the cardioprotective effects of celastrol pretreatment on I/R injury and to further explore whether its mechanism of action was associated with the inhibition of high mobility group box 1 protein (HMGB1) expression via the phosphoinositide 3-kinase (PI3K)/Akt pathway. In a fixed-dose study, hematoxylin and eosin staining and myocardial enzyme measurements were used to determine the optimal dose of celastrol that elicited the best cardioprotective effects against I/R injury. Furthermore, rats were pretreated with 4 mg/kg celastrol, and infarct size and the levels of myocardial enzymes, apoptosis, inflammatory and oxidative indices, and HMGB1 and p-Akt expression were measured. Our results indicated that celastrol dose-dependently attenuated histopathological changes and the elevation in myocardial enzymes induced by I/R. Moreover, the celastrol pretreatment (4 mg/kg) not only significantly decreased infarct size as well as myocardial enzyme levels but also inhibited myocardial apoptosis, inflammatory response and oxidative stress. Additionally, celastrol downregulated HMGB1 expression and upregulated p-Akt expression in the myocardium. LY294002, a specific pI3k inhibitor, partially reversed the decreased HMGB1 expression, increased p-Akt expression induced by celastrol, and abolished the anti-apoptotic, anti-inflammatory and anti-oxidative effects of celastrol. These findings suggest that short-term pretreatment with celastrol protects against myocardial I/R injury by suppressing myocardial apoptosis, inflammatory response and oxidative stress via pI3k/Akt pathway activation and HMGB1 inhibition.